Adenovirus (Ad) DNA synthesis will be studied using a recently developed system that initiates and elongates Ad templates in vitro. Replication depends on 5 purified components. Three are viral coded: the DNA polymerase (Pol), a precursor to the covalently-linked terminal protein (pTP) and the DNA binding protein (DBP). Two additional replication factors (I and II) are present in uninfected nuclear extracts. Factor II is a DNA topoisomerase. An understanding of the control of DNA replication is critical to an understanding of several important disease states including malignancies. Accordingly, this in vitro DNA replication system which is unique among eukaryocytes in that it can initiate new DNA strands will be used to study: (1) functionally active domains of the DNA Pol and pTP. Genes for these 2 viral coded proteins will be cloned and expressed in prokaryotic vectors. Site specific mutagenesis will allow the active domains of pTP and Pol to be mapped. (2) Temperature sensitive mutations in the Ad DBP, are correctable by second site mutations which require an RNA cofactor for full activity. The nature of the RNA will be studied. (3) Viral core proteins inhibit the in vitro DNA synthesis reaction and the mechanism of this inhibition will be studied. (4) The effect of several components (VP-16-213, VM26 and camptothecin), that inhibit Ad DNA synthesis by cleaving the DNA template can now be studied in vitro for all steps of the breakage reaction can be reproduced in such systems. (5) Studies will be continued on inhibition of Ad DNA elongation by antibody from patients with systemic lupus erythematosus. In addition to the inhibition already shown by sera with antibody to double-stranded DNA, antibodies to nuclear factors I and II will be specifically sought.